Evaluation of the metal-dependent cytotoxic behaviour of coordination compounds

Article abstract of DOI:10.1039/c7dt04604a

The [Cu(L)Cl₂]₂ and [Pt(L)Cl₂] complexes were prepared from the simple Schiff-base ligand (E)-phenyl-N-((pyridin-2-yl)methylene)methanamine (L) and respectively, CuCl₂ and cis-[PtCl₂(DMSO)₂]. DNA-interaction studies revealed that the copper complex most likely acts as a DNA cleaver whereas the platinum complex binds to the double helix. Remarkably, cell-viability experiments with HeLa, MCF7 and PC3 cells showed that [Cu(L)Cl₂]₂ is an efficient cytotoxic agent whereas [Pt(L)Cl₂] is not toxic, illustrating the crucial role played by the nature of the metal ion in the corresponding biological activity.

Full text of DOI:10.1039/c7dt04604a

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ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
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PAPER
Evaluation of the metal-dependent cytotoxic behaviour of
coordination compounds
Jordi Grau,a Cristina Renau,a Ana B. Caballero,a Amparo Caubet,*a Marta Pockaj,b Julia Lorenzoc
and Patrick Gamez*ade
Received 00th January 2018,
Accepted 00th January 2018
DOI: 10.1039/x0xx00000x
The [Cu(L)Cl2]2 and [Pt(L)Cl2] complexes were prepared from the simple Schiff-base ligand (E)-phenyl-N-((pyridin-2-
yl)methylene)methanamine (L) and respectively, CuCl2 and cis-[PtCl2(DMSO)2]. DNA-interaction studies revealed that the
copper complex most likely acts as a DNA cleaver whereas the platinum complex binds to the double helix. Remarkably,
cell-viability experiments with HeLa, MCF7 and PC3 cells showed that [Cu(L)Cl2]2 is an efficient cytotoxic agent whereas
[Pt(L)Cl2] is not toxic, illustrating the crucial role played by the nature of the metal ion on the corresponding biological
activity.
www.rsc.org/
context, copper represents a good alternative as it is an
essential trace element present in the human body (it is
therefore biocompatible). Moreover, copper is naturally more
Introduction
Since the fortuitous discovery of the anticancer properties of
abundant than platinum, and is thus far cheaper than the
cis-diamminedichloridoplatinum(II), cisplatin, by Rosenberg
2
and co-workers in the 1960s,1, there has been growing
precious metal. Actually, since the initial work of Sigman and
13
4
co-workers in the 1970s,12,
the scientific community has
interest in the design of platinum-based drugs,3, and more
shown increasing interest in copper complexes.14 For instance,
some mixed-chelate copper complexes of the Casiopeínas®
family have entered clinical trials.15
generally of metal complexes as potential chemotherapeutic
agents.5
Coordination compounds offer high versatility for drug
design; indeed, besides the nature of the metal and its
oxidation state, various geometries and coordination numbers
may be adopted by metal ions, which allow to fine-tune their
chemical reactivity through the adjustment of kinetic (rates of
ligand exchange) and/or thermodynamic (metal–ligand bond
strengths, redox potentials, etc.) parameters.6 The ligands may
also be involved in the observed biological activity of the
complexes, through (i) their participation in the outer-sphere
recognition of the target site,7 (ii) the (cytotoxic) activity of any
released ligands8 or (iii) ligand-centred redox processes.9
In the present study, a very simple, readily available Schiff-
base
ligand,
namely
(E)-phenyl-N-((pyridin-2-
yl)methylene)methanamine16, 17
(L
; Figure 1), has been used to
generate two different coordination compounds from the
metal chloride salts cis-[PtCl2(DMSO)2] and CuCl2. Comparison
of the DNA-interacting and cytotoxic behaviours of the
complexes obtained, i.e. [Pt(L)Cl2] and [Cu(L)Cl2]2, reveals that
the nature of the metal ion has a drastic effect on the
biological properties of the corresponding
compound.
“[M(L)Cl2]”
Apart from platinum, other transition-metal ions are used
11
In that
to develop potential anticancer metallodrugs.10,
Experimental
Materials and methods
Reagents and solvents were obtained from Sigma-Aldrich or
Fisher Scientific and used as received. pBR322 plasmid DNA
and calf-thymus DNA (ct-DNA) were purchased from Sigma-
Aldrich.
Nuclear magnetic resonance (1H and 13C{1H}) spectra were
recorded at room temperature on a Varian Unity 400 MHz
spectrometer. Proton chemical shifts are expressed in parts
per million (ppm, δ scale) and are referenced to residual
solvent peaks. Infrared (IR) spectra (KBr pellets) were recorded
with a Nicolet-5700 FT-IR (in the range 4000–400 cm−1), and
data are represented as the frequency of absorption in cm−1.
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Elemental analyses were carried out by the Serveis Científic i left unperturbed for the slow evaporation of the solvent.
Tecnològics de la Universitat de Barcelona. UV-Vis experiments Green single crystals, suitable for X-ray diffraction
were performed with a Varian Cary-100 spectrophotometer, measurements (see Supporting Information for X-ray data),
and fluorescence measurements were achieved using
a
were obtained with a yield of 51% (based on copper).
HORIBA Jobin–Yvon iHR320 spectrofluorimeter at room Elemental analysis calcd. for C26H24Cl4N4Cu2 (%): C 47.21, H
temperature. The photomultiplier detector voltage was set at 3.66, N 8.47; found: C 47.38, H 3.89, N 8.58. IR (KBr, cm–1):
950 V, and the instrument excitation and emission slits were 3439 (ν(OH) + ν(NH)), 3065 (ν(Csp2H)), 1608 (ν(C=N)), 1062 (ν(C-
both set at 5 nm. The concentrations of DNA and complexes N)).
used for the UV-vis and fluorescence studies are described in
Gel Electrophoresis.
the section Results and Discussion.
[Cu(L)Cl2]2: a stock solution of the complex was prepared in
Preparation of the ligand L and the complexes [[Pt(L)Cl2] and
1 mM sodium cacodylate–20 mM NaCl buffer (pH = 7.2).
pBR322 plasmid DNA aliquots (0.01 mg mL−1, 15 µM referred
[Cu(L)Cl2]2.
Ligand:
The
ligand
(E)-phenyl-N-((pyridin-2- to base pairs) in 1 mM cacodylate–20 mM NaCl buffer were
yl)methylene)methanamine16 (Scheme 1,
L
) was prepared by incubated with [Cu(L)Cl2]2 for 1 h at 37 °C. Subsequently,
ascorbic acid (1 mM cacodylate–20 mM NaCl buffer) was
added (in the case of the experiments without ascorbic acid,
this step was not done), and the resulting mixture was
incubated at 37 °C for an additional hour. Next, the reaction
samples were quenched with 4 μL of a solution containing 30%
(v/v) glycerol and 0.25% (w/v) xylene cyanol, and then
electrophoretized on agarose gel (1% in TAE buffer, Tris-
acetate–EDTA) for 2 h at 1.5 V cm−1. The electrophoresis was
carried out using a BIORAD horizontal tank connected to a
CONSORT EV231 variable potential power supply. Samples of
free DNA and DNA in the presence of ascorbic acid were used
as controls. Afterwards, the DNA was stained with SYBR® safe
and the gel was photographed with a BIORAD Gel Doc™ EZ
Imager.
condensation of pyridine-2-carbaldehyde and benzylamine.
was obtained as an oil, with a yield of 70%.
L
1H NMR (400 MHz, CDCl3): δ = 8.62 (d, 1H, H6), 8.47 (s, 1H,
H7), 8.05 (d, 1H, H3), 7.71 (t, 1H, H4), 7.27 (m, 2H, H11 and
H12), 7.23 (t, 1H, H5), 7.20 (t, 1H, H13), 4.86 (s, 2H, H9).
13C{1H}-NMR (101 MHz, CDCl3, ppm): δ = 164.3 (C7), 156.0 (C2),
150.8 (C6), 140.1 (C10), 138.1 (C4), 130.0 (C12), 129.7 (C11),
128.7 (C13), 126.3 (C5), 122.8 (C3), 66.4 (C9). IR (KBr, cm–1):
3383 (ν(NH)), 3100-3000 (ν(Csp2H)), 1643 (ν(C=N)), 1188 (ν(C-
N)).
[Pt(L)Cl2]: Solutions containing 10-100 uM of the complex, 1 %
DMSO and 15 µMbp pBR322 plasmid DNA were incubated for 24 h
[Pt(L)Cl2]: a methanolic solution of cis-[PtCl2(DMSO)2] (0.211 g; 0.5
mmol; 20 mL) was refluxed until dissolution of the platinum
precursor. After filtration, a methanolic solution of ligand L (0.106 g;
0.5 mmol; 3 mL) was added and the resulting reaction mixture was
stirred at room temperature for 30 minutes. Subsequently, the
orange precipitate was isolated by filtration and washed with
methanol. After drying in air, 0.091 g of the platinum(II) complex
at 37 °C. Next, the reaction samples were quenched with 4 μL
of a solution containing 30% (v/v) glycerol and 0.25% (w/v)
xylene cyanol, and then electrophoretized on agarose gel (1%
in TBE buffer 1X, Tris-Borate–EDTA) for 1 h at 6.25 V cm−1
.
X-ray crystallography. Single-crystal X-ray diffraction.
A single crystal of [Cu(L)Cl2]2 in silicon grease was mounted on
the tip of a glass fibre and transferred to the goniometer head,
under a cryostream of nitrogen. Data were collected on a
SuperNova diffractometer equipped with an Atlas detector,
using CrysAlis PRO software, and monochromated Mo K
α
radiation (0.71073 Å) at 150 K.18 The initial structural model
containing the coordination molecule was obtained using the
Superflip structure solution program.19 Full-matrix least-
squares refinement on F2 with anisotropic displacement
parameters for all nonhydrogen atoms was carried out using
SHELXL2017.20 All H atoms were initially located in difference
Fourier maps and were further treated as riding on their
was obtained (Yield
= 39%). Elemental analysis calcd. for
C13H12Cl2N2Pt (%): C 33.78, H 2.62, N 6.06; found: C 33.62, H
2.63, N 6.06. 1H NMR (400 MHz, [D6]DMSO): δ = 9.37 (d, 1H,
H2), 9.30 (s, 1H, H7), 8.37 (t, 1H, H4), 8.16 (d, 1H, H5), 7.91 (t,
1H, H3), 7.53 (d, 1H, H11), 7.40 (t, 1H, H12), 7.35 (t, 1H, H13),
5.32 (s, 2H, H9). 13C{1H}-NMR (101 MHz, [D6]DMSO, ppm): δ =
171.9 (C7), 156.9 (C6), 148.8 (C2), 140.5 (C4), 138.2 (C10),
136.0 (C3), 129.2 (C5), 128.8 (C11), 128.4 (C12), 128.0 (C13),
60.9 (C9). IR (KBr, cm–1): 3100-3000 (ν(Csp2H)), 1622 (ν(C=N)),
522 and 448 (ν(Pt-N)).
parent atoms with C(aromatic)−H
=
0.95
Å
and
C(methylene)−H = 0.99 Å. Details on crystal data, data
collection and structure refinement are given in the Supporting
Information.
These data can be obtained free of charge from the Cambridge
[Cu(L)Cl2]2: An ethanolic solution of ligand
L (39.3 mg, 0.2
Crystallographic
Data
Center
via
mmol; 5 mL) was added to an ethanolic solution of CuCl2∙2H2O
(34.0 mg, 0.2 mmol; 15 mL). The resulting green solution was
https://summary.ccdc.cam.ac.uk/structure-summary-form.
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Cell-line culture and cell viability assays
(see Figure S1). However, since one of the Cl anions is semi-
coordinated to the Cu ion (Cu−Cl distance superior to 2.6 Å;
see Table S2), it can be expected that the bis-μ-Cl bridge is
broken in solution,22 generating a mononuclear species.
The interaction of the two complexes with DNA was
subsequently examined using different techniques.
The cancer cells (HeLa, MCF7 and PC3 cell lines) were cultured
in Minimum Essential Medium (MEM) alpha medium
supplemented with 10% (v/v) heat inactivated fetal bovine
serum (FBS) in a highly humidified atmosphere of 95% air with
5% C02 at 37 °C.
Cytotoxicity was determined using PrestoBlue cell viability
assay (ThermoFisher). The cells were seeded in a 96-well
microtiter plate at a concentration of 1 × 104 cells per well and
incubated overnight in an atmosphere containing 5% CO2 at
37 °C. The cells were exposed to the positive drug control
UV-vis studies. The interaction of [Cu(L)Cl2]2 and [Pt(L)Cl2] with
DNA was first investigated by UV-vis spectrophotometric titration.
Addition of increasing amounts of ct-DNA (from 0 to 100 µM, in
base pairs) to a 25 µM solution of [Cu(L)Cl2]2 resulted in clear
hypochromicity but no absorption shift was observed (Figure
2). These features suggest that [Cu(L)Cl2]2 interacts outside the
minor groove of DNA.23 The corresponding Kb value of (1.4 ±
0.2) 105 M─1 was determined by plotting [DNA]/(εf─εa) versus
[DNA], εf being the extinction coefficient of the free complex in
solution (Aobs/[complex]) and εa the extinction coefficient of
the DNA-bound complex (insert in Figure 2).
cisplatin, to the free ligand L, and to complexes [Pt(L)Cl2]2 and
[Cu(L)Cl2]2. The microtiter plate was incubated for 24 h or 72ꢀh,
and 20ꢀµL of PrestoBlue were subsequently added. The plates
were incubated for 2ꢀadditional hours, and the fluorescence
was measured exciting at 531 nm and 570ꢀnm, using a Victor3
V multiwell plate reader (Perkin Elmer). The cytotoxicity of
each sample was expressed as the IC50 value, which is the
concentration of the complex that caused 50% inhibition of
cell growth. All the samples were assayed in triplicate.
For [Pt(L)Cl2], hypochromicity with a slight red shift (∆λ = 2
nm) of the absorption is observed (Figure 3). The small
bathochromic effect associated with the decrease of the
absorption intensity can be ascribed to intercalation, but
outside binding cannot be excluded (indeed, strong
intercalation is characterized by a large red shift, of more than
The induction of apoptosis in vitro by the different complexes
was determined with HeLa cells by a flow cytometric assay
with Annexin V-FITC, using the Annexin V-FITC apoptosis
detection kit from Roche. Exponentially growing HeLa cells in
6-well plates (4 × 105 cells per well) were exposed to
concentrations equal to the IC50 values at 24 h of the
complexes, or cisplatin as a reference. The cells were collected,
washed with PBS, and resuspended in 100 μL of binding buffer.
2 μL of Annexin V-fluorescein isothiocyanate (FITC) and 2 μL of
propidium iodide (PI) were added to the samples for 15 min at
room temperature in the dark. The number of apoptotic cells
was analysed by flow cytometry (FacsCalibur, Becton
Dickinson).
10 nm).24 These data indicate that [Pt(L)Cl2
] most likely
interacts with DNA via different binding modes. The
determined Kb value of (7.2 ± 0.1) × 106 M─1 illustrates a
stronger DNA interaction of [Pt(L)Cl2], compared with that of
[Cu(L)Cl2]2
.
Fluorescence studies. The ability of [Cu(L)Cl2]2 to displace the
DNA-intercalator ethidium bromide (EB) was evaluated using
fluorescence spectroscopy. Increasing amounts of the copper
compound (0─100 μM) have been added to a solution of the
fluorescent EB-DNA complex (constant concentrations of EB
and ct-DNA of 25.36 mM and 25 μM, respectively). A
moderate decrease in emission intensity is achieved (Figure 4),
which also indicates that [Cu(L)Cl2]2 is not acting as an
intercalator (see UV-vis data). It can be pointed out here that
the poor displacement of EB observed may indeed be
explained by electrostatic interactions or groove binding; such
interactions can be sufficient to modify the conformation of
the DNA double helix, resulting in the release of EB.25, 26 The
corresponding Stern-Volmer quenching constant (KSV),
determined by plotting I0/I versus [complex] (I0 and I being the
emission intensities in the absence and the presence of the
complex, respectively),27 is (1.9 ± 0.1) × 103 M‒1. This value
illustrates poor intercalating properties for the copper
compound, as already inferred from the UV-vis studies (see
above); most likely, [Cu(L)Cl2]2 acts as a groove binder.
Results and discussion
The
ligand
(E)-phenyl-N-((pyridin-2-
yl)methylene)methanamine (
L
) was obtained in good yield by
simple condensation reaction between benzylamine and 2-
pyridinecarboxaldehyde, in ethanol under reflux. The
complexes were prepared by mixing equivalent amounts of
ligand
L and the respective metal-chloride precursor in an
alcoholic solvent (see Experimental Section). [Pt(L)Cl2] was
obtained in methanol with a yield of 39% while [Cu(L)Cl2]2 was
isolated from an ethanolic solution with a yield of 51%. Single
crystals of the copper complex were obtained, whose X-ray
diffraction analysis confirmed the structure previously
described in the literature (Figure S1).21 Crystallographic data
for [Cu(L)Cl2]2 are summarized in Table S1, and selected
coordination bond lengths and angles are listed in Table S2. It
is important to notice that the two metal complexes display
similar coordination environments, which is illustrated in
Figure 1. In both cases, a square plane is present, formed
In the case of the platinum complex, a non-linear I0/I vs.
[complex] plot is obtained (Figure 5). As for the UV-vis study
(see above), the fluorescence data also indicate that [Pt(L)Cl2
]
interacts through various modes of binding with the double
helix. The Stern-Volmer constant was determined for the initial
points of the plot, namely for complex concentrations of 0, 5,
10 and 15 μM (insert of Figure 5). The obtained KSV of (7.5 ±
through the cis-coordination of a chelating
L ligand and two
chlorides to the metal center. It can be stressed that, in the
solid state, the copper complex is a chlorido-bridged dimer
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0.5) × 103 M‒1 suggests that the platinum compound has some the plasmid upon platinum binding. A higher complex
DNA-intercalating behaviour at low [complex]. For [complex] > concentration, namely 50 μM (3.33 equivalents), shows a
15 μM, no release of EB is observed, which may be explained different behaviour (lane 6); indeed, a combination between a
by another mode of interaction, for instance groove binding cisplatin-like effect and single-stand break is observed,
(as corroborated by the UV-vis data; see above).
suggesting that the binding of the complex eventually leads to
the cleavage of the biomolecule. Actually, at a concentration
Gel electrophoresis. The interaction of [Cu(L)Cl2]2 with DNA of 100 μM (6.67 equivalents), complete relaxation of
was directly observed with agarose gel electrophoresis. supercoiled DNA (form I) is observed, and the sole presence of
Electrophoretic mobility measurements of pBR322 plasmid form II is detected. tend
DNA were therefore carried out using increasing amounts of
the copper complex (5─100 μM). Ascorbic acid was used as Cytotoxicity assays. Cytotoxicity studies were carried out for
reducing agent, which mimics the reducing environment found both compounds with HeLa (cervical carcinoma),32 MCF7
inside cells. The resulting generation of copper(I) species is (breast adenocarcinoma) and PC3 (prostatic small cell
thus expected to mediate the formation of reactive oxygen carcinoma) cells. The aim was to illustrate that copper
species that will cleave DNA. The consequent agarose gel is compounds may offer an alternative to platinum drugs. The
depicted in Figure 6a. As expected, pure plasmid DNA ([DNA] = cytotoxic behaviour of both complexes was thus investigated
15 μM in base pairs) gives two bands (lane 1), the most intense and compared with that of the free ligand L, copper(II) chloride
one corresponding to supercoiled DNA (form I), while the and cisplatin. The IC50 values obtained after 24 and 72 hours of
second one is ascribed to the circular nicked form (form II). In incubation with the three different cell lines are listed in
the absence of ascorbic acid (lanes 2─8), [Cu(L)Cl2]2 does not Table 1.
affect the structure of the biomolecule. Similarly, the well-
Table 1 IC50 values (µM) for the two complexes, the free ligand L, copper(II) chloride
known DNA cleaver [Cu(phen)2]2+ (which is the copper-based
and cisplatin against HeLa, MCF7 and PC3 cells, after 24 and 72 h incubation.
chemical nuclease of reference)28 is not active with no added
HeLa
24 h
reducing agent (lane 9; [complex] = 40 μM). In contrast, when
ascorbic acid is added to [Cu(phen)2]2+,
complete
Complex
[Pt( )Cl2]
[Cu( )Cl2]2
72 h
73.9 ± 4.1
7.7 ± 1.4
n.a.a
a
L
> 100
degradation of DNA is observed (lane 10). It can be noticed
that in the sole presence of the reducing agent, i.e. without
the copper compound, the DNA is not altered (lane 11). For
[Cu(L)Cl2]2, as for the copper-phenanthroline complex, the
presence of reducing agent gives rise to DNA cleavage (lanes
12─19). Already at a complex concentration of 5 μM, an
increase in form II is noticed (form II is obtained when one of
the two strands are cut apart), which is associated to a
decrease in form I (lane 12). At a concentration of 20 μM, form
I has completely disappeared and the linear form, i.e. form III
resulting from the breakage of both strands, is observed (lane
L
8.5 ± 1.3
n.a.a
n.a.a
Ligand
CuCl2
L
> 200
cisplatin
40.3 ± 3.7
14.7 ± 2.6
MCF7
> 100
[Pt(
L)Cl2]
45.5 ± 6.1
5.1 ± 1.1
n.a.a
[Cu(
L)Cl2]2
13.6 ± 1.2
n.a.a
n.a.a
Ligand
CuCl2
L
> 200
cisplatin
38.3 ± 3.2
5.6 ± 0.4
15). From
a complex concentration of 40 μM, total
PC3
> 100
degradation of DNA is achieved (lanes 16─19). Hence,
[Cu(L)Cl2]2 appears to be at least as active as the reference
compound [Cu(phen)2]2+ (see lanes 10 and 16). Thus, although
[Cu(L)Cl2]2 is not strongly interacting with DNA (see UV-vis and
fluorescence data above), most likely it induces the formation
of radical species (such as Sigman’s reagent, i.e.
[Cu(phen)2]2+)29 that are really harmful to the biomolecule.
The gel electrophoresis results for [Pt(L)Cl2] are shown in
Figure 6b. Incubation of DNA with 0.5 equivalents of cisplatin
affects the electrophoretic mobility of both form I and form II;
cisplatin-mediated unwinding of form I causes a diminution of
its mobility, whereas the unwinding of form II increases its
mobility because of a shortening effect on the DNA contour
[Pt(
L
)Cl2]
78.8 ± 8.8
9.7 ± 1.1
n.a.a
[Cu(
L
)Cl2]2
17.2 ± 2.9
n.a.a
n.a.a
Ligand
CuCl2
L
> 200
cisplatin
49.8 ± 5.4
7.4 ± 0.9
a
n.a. stands for ´not active´ and indicates that IC50 values could not be obtained
(due to non-cytotoxicity).
The copper complex [Cu(L)Cl2]2 exhibits very good cytotoxic
properties; after 24 h incubation with HeLa cells, an IC50 value
of 8.5 ± 1.3 μM is reached (Table 1). In contrast, the platinum
compound [Pt(L)Cl2] is not active. After 72 hours of incubation,
[Cu(L)Cl2]2 is 10 times more active than [Pt(L)Cl2]; i.e. 7.7 ± 1.4
length (see lanes 1 and 2).30, These effects become even
31
versus 73.9 ± 4.1 μM, respectively. The free ligand
L is not
more pronounced when the concentration of cisplatin is
increased, up to the point that the two bands coalesce; in fact,
a single broad band is observed when 1.0 equivalent is used
cytotoxic so as copper(II) chloride. Cisplatin shows some
moderate activity against this line, albeit superior to that
observed for [Pt(L)Cl2]. With MCF7 cells, the same tendency is
observed, the copper compound being 9 times more active
than the platinum one (i.e. 5.1 ± 1.1 versus 45.5 ± 6.1 μM). The
(lane 3). 0.67 and 1.67 equivalents of [Pt(L)Cl2] appear to
induce the unwinding of form I, converting it into form II (lanes
4 and 5). This may be explained by single-strand cleavage of
4 | Dalton Trans., 2018, 47, 1-6
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higher toxicity of [Cu(L)Cl2]2 is further confirmed using PC3 action) but can result from oxidative stress (Sigman-like mode
cells; after 72 h incubation, its IC50 value for this cell line is 9.7 of action).
± 1.1 while it is 78.8 ± 8.8 for [Pt(L)Cl2]. The cytotoxic activities
of [Cu(L)Cl2]2 against MCF7 and PC3 cells are comparable to have been prepared from the same organic ligand, namely (E)-
that of cisplatin. phenyl-N-((pyridin-2-yl)methylene)methanamine, and
In the present study, a copper and a platinum complexes
In all cases, [Cu(L)Cl2]2 is clearly the most active compound, inorganic counter-ions, viz. chloride anions. The DNA-
which illustrates the crucial role played by the metal ion. interacting properties of the two related compounds (differing
Indeed, [Cu(L)Cl2]2 and [Pt(L)Cl2] have similar coordination by their metal centre) were examined, and their cytotoxicity
environments built up from the same ligands, namely chloride behaviour was compared using HeLa, MCF7 and PC3 cells.
anions and the simple bidentate ligand (E)-phenyl-N-((pyridin-
2-yl)methylene)methanamine (ligand ). Remarkably, is non- compounds appear to affect DNA, but with different
cytotoxic and [Pt(L)Cl2] is poorly cytotoxic, whereas [Cu(L)Cl2]2 mechanisms of action. While the Cu complex clearly induces
displays an interesting behaviour (Table 1). DNA cleavage, acting like Sigman’s reagent, the platinum
The results achieved are very instructive; indeed, the two
L
L
In summary, although [Pt(L)Cl2] appears to better interact complex exhibits a behaviour that is analogous to that of
with DNA than [Cu(L)Cl2]2 (see above), it exhibits significantly cisplatin. Nevertheless, the platinum complex is poorly
lower cytotoxicities than the copper compound. Clearly, the cytotoxic (i.e., it is not active after 24 h incubation), whereas
formation of reactive oxygen species (ROS) mediated by the the copper complex is significantly cytotoxic (IC50 values in the
redox-active copper complex is crucial regarding cell toxicity.
range 8.5-17.2 μM after 24 h incubation). Remarkably, the free
Subsequently, the ability of [Cu(L)Cl2]2 to induce apoptosis ligand nor copper(II) chloride show cytotoxicity, therefore
was examined and compared with that of cisplatin, using HeLa illustrating the crucial effect of the association copper-ligand
cells. The experiments were carried out using an incubation on the biological activity. Moreover, the role played by the
time of 24 hours, and complex concentrations corresponding nature of the coordinated metal ion is key as the same ligand
to the IC50 value for each compound at this incubation period leads to metal complexes with drastically distinct behaviours.
(see Table 1; HeLa cells). The results obtained are shown in
Table 2.
As recently mentioned in a
review paper,29 more
systematic studies are needed to appraise the potential of
copper complexes as anti-cancer agents to substitute platinum
drugs; herein, it has been shown that a ligand generating a
non-active Pt complex produces an active Cu complex, which
induces cell apoptosis. These results hence indicate that metal
screening with a ligand is important as it may allow to identify
coordination compounds with interesting properties.
Table 2 Percentage of HeLa cells in each state after 24 hours of incubation, in the
absence (control) and presence of IC50 concentrations (see Table 1; 24 h) of [Cu(L)Cl2]2
and cisplatin.
Treatment
(IC50, 24 h)
control
% vital
cells (R1)
90.5
% apoptotic
cells (R2)
4.7
% late apoptosis
% damaged
cells (R4)
1.6
or dead cells (R3)
3.3
[Cu(
L
)Cl2]2
68.9
16.6
13.1
13.4
1.3
cisplatin
62.2
23.9
0.5
Acknowledgements
Financial support from the Spanish Ministerio de Economía y
Competitividad (projects CTQ2015-70371-REDT, CTQ2014-
55293-P and CTQ2017-88446-R) is acknowledged. PG
acknowledges the Institució Catalana de Recerca i Estudis
Avançats (ICREA).
Interestingly, the copper complex induces apoptosis of
HeLa cells, albeit in a slightly less efficient manner than
cisplatin (17% versus 24%; Table 2). Thus, it appears that
[Cu(L)Cl2]2 causes apoptotic cell death through a mechanism of
action, i.e. production of ROS, which is different to that of the
well-known platinum compound.
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Figure 1. Schematic illustration of the coordination environment of the metal
centres, showing the formation of a square plane from a cis N2 ligand ( ) and to
chlorides.
Figure 4. Emission spectra of the EB-DNA complex ([c] = 25 μM), λexc = 514 nm,
λem = 610 nm, upon addition of increasing amounts of [Cu(L)Cl2]2 (0−200 μM).
The blue arrow shows the diminution in emission intensity with the increase of
[complex]. Insert: I0/I vs. [complex] plot for the titration of EB-DNA with
[Cu(L)Cl2]2; experimental data point and linear fitting of the data.
L
Figure 2. Absorption spectra of [Cu(L)Cl2]2 upon addition of ct-DNA. Complex
concentration = 25 μM; [DNA] = 0−100 μM (DNA concentration determined at λ
= 260 nm, with ε = 6600 M−1 cm−1). The MLCT band at λ = 288 nm was used to
determine Kb (insert). The blue arrows show the decrease in absorption intensity
with the increase of [ct-DNA].
Figure 5. Plot of I0/I vs. [complex] for the titration of EB-DNA with [Pt(L)Cl2] at λexc
= 514 nm and λem = 610 nm; experimental data point and fitting of the data.
[complex] = 0−150 μM; [ct-DNA] = 25 μM and [EB] = 125 μM. Insert: linear fitting
of the data for [complex] = 0−15 μM.
Figure 3. Absorption spectra of [Pt(L)Cl2] upon addition of ct-DNA. Complex
concentration = 25 μM; [DNA] = 0−100 μM (DNA concentration determined at λ
= 260 nm, with ε = 6600 M−1 cm−1). The MLCT band at λ = 257-259 nm was used
to determine Kb (insert). The blue arrow shows (i) the decrease in absorption
intensity with the increase of [ct-DNA] and (ii) the slight red shift of the
absorption upon addition of ct-DNA.
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Figure 6. Agarose gel electrophoresis images of pBR322 plasmid DNA incubated
for 24 h at 37 °C with increasing concentrations of a) [Cu(L)Cl2]2 and b) [Pt(L)Cl2]
.
a) Lane 1: pure plasmid DNA; lanes 2−8: without reducing agent – lane 2:
[complex] = 5 μM, lane 3: [complex] = 10 μM, lane 4: [complex] = 20 μM, lane 5:
[complex] = 40 μM, lane 6: [complex] = 60 μM, lane 7: [complex] = 80 μM, lane 8,
[complex] = 100 μM − lane 9: [Sigman’s reagent] = 40 μM; lane 10: [Sigman’s
reagent] = 40 μM + [ascorbic acid] = 1 mM; lane 11: pure plasmid DNA; lane 12:
DNA + [ascorbic acid] = 1 mM; lanes 13-19: with reducing agent ([ascorbic acid] =
1 mM) – lane 13: [complex] = 5 μM, lane 14: [complex] = 10 μM, lane 15:
[complex] = 20 μM, lane 16: [complex] = 40 μM, lane 17: [complex] = 60 μM,
lane 18: [complex] = 80 μM, lane 19, [complex] = 100 μM.
b) Lane 1: pure plasmid DNA; lane 2: DNA + cisplatin (0.5 equiv.); lane 3: DNA +
cisplatin (1.0 equiv.); lane 4: [complex] = 10 μM; lane 5: [complex] = 15 μM; lane
6: [complex] = 50 μM; lane 7: [complex] = 100 μM.
8 | Dalton Trans., 2018, 47, 1-6
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